Anti-inflammatory method using gamma-aminobutyric acid (GABA) analogs

ABSTRACT

GABA analogs such as gabapentin and pregabalin are useful to prevent and treat inflammatory diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/403,867 filed Oct.25, 1999, now U.S. Pat. No. 6,329,429, which is a §371 of PCT/US98/13107filed Jun. 24, 1998, and claims benefit of priority from U.S.Provisional Application Nos. 60/050,736 filed Jun. 25, 1997, and60/084,183 filed May 4, 1998.

This invention was made in part with United States Government supportunder Grant No. IR01NS32778-01A1 administered by the National Instituteof Health. The Federal Government may own certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates to a method for treating inflammatory diseases byadministering a gamma-aminobutyric acid (GABA) analog.

BACKGROUND OF THE INVENTION

Inflammatory diseases are characterized by a complex series ofhistological events, including dilatation of arterioles, capillaries,and venules, with increased permeability and blood flow; exudation offluids, including plasma proteins; and leukocytic migration into theinflammatory focus. Many forms of inflammation are localized protectiveresponses elicited by injury or destruction of tissues, which serves todestroy, dilute, or wall off both the injurious agent and the injuredtissue. The inflammatory response itself is also responsible forpathologic tissue damage. Arthritis is a particularly devastatinginflammatory disease, generally affecting older people, and ischaracterized by the inflammatory lesions being primarily confined toarticular joints. The disease is marked by pain, heat, redness,swelling, and tissue destruction. Rheumatoid arthritis is a chronicsystemic disease of the joints, marked by inflammatory changes in thesynovial tissue and articular structures, and by atrophy and rarefactionof the bones. This form of inflammatory disease generally progresses todeformity and ankylosis.

Numerous anti-inflammatory treatments are known and commonly used. Themost common are the nonsteroidal anti-inflammatory agents such asnaproxen, diflunisal, mefenamic acid, and ketorolac tromethamine. Theseagents generally are used to treat short term mild inflammation andpain. More severe inflammatory disease, such as arthritis, are treatedwith steroidal hormones and glucocorticoids, for example prednisolone,hydrocortisone acetate, and betamethasone sodium phosphate.

Because many of the anti-inflammatory agents are only short acting, andoften produce severe side effects, the need for new therapies continue.We have now discovered that compounds which are analogs of gammaaminobutyric acid (GABA) are useful to treat inflammatory diseases. Allthat is required to prevent or treat the inflammatory disease accordingto this invention is to administer to a subject in need of treatment ananti-inflammatory amount of a GABA analog.

Several GABA analogs are known. Gabapentin, a cyclic GABA analog, is nowcommercially available and extensively used clinically for treatment ofepilepsy and neuropathic pain. Such compounds are described in U.S. Pat.No. 4,024,175. Another series of GABA analogs is described in U.S. Pat.No. 5,563,175.

SUMMARY OF THE INVENTION

This invention provides a method for preventing and treatinginflammatory diseases comprising administering to a subject sufferingfrom such disease or suspected of developing such disease and in need oftreatment an effective amount of a GABA analog. A preferred embodimentutilizes a cyclic amino acid compound of Formula I

wherein R₁ is hydrogen or lower alkyl and n is an integer of from 4 to6, and the pharmaceutically acceptable salts thereof. An especiallypreferred embodiment utilizes a compound of Formula I where R₁ ishydrogen and n is 5, which compound is 1-(aminomethyl)-cyclohexaneacetic acid, known generically as gabapentin. Other preferred GABAanalogs have Formula I wherein the cyclic ring is substituted, forexample with alkyl such as methyl or ethyl. Typical of such compoundsinclude (1-aminomethyl-3-methylcyclohexyl) acetic acid,(1-aminomethyl-3-methylcyclopentyl) acetic acid, and(1-aminomethyl-3,4-dimethylcyclopentyl) acetic acid.

In another embodiment, the anti-inflammatory method of the inventionutilizes a GABA analog of Formula II

or a pharmaceutically acceptable salt thereof, wherein

-   R₁ is a straight or branched alkyl of from 1 to 6 carbon atoms,    phenyl, or cycloalkyl of from 3 to 6 carbon atoms;-   R₂ is hydrogen or methyl; and-   R₃ is hydrogen, methyl, or carboxyl.

Diastereomers and enantiomers of compounds of Formula II can be utilizedin the invention.

An especially preferred method of the invention employs a compound whereR₂ and R₃ are both hydrogen, and R₁ is —(CH₂)₀₋₂—i C₄H₉ as an (R), (S),or (R,S) isomer.

A more preferred embodiment of the invention utilizes3-aminomethyl-5-methyl-hexanoic acid, and especially(S)-3-(aminomethyl)-5-methylhexanoic acid, now known generically aspregabalin. Pregabalin is also known as “CI-1008” and “S-(+)-3-IBG.”Another preferred compound of Formula II is3-(1-aminoethyl)-5-methylhepanoic acid.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the method of this invention utilizes any GABA analog. AGABA analog is any compound derived from or based upongamma-aminobutyric acid, and causes an anti-inflammatory effect inaccordance with this invention. The compounds are readily available,either commercially, or by synthetic methodology well-known to thoseskilled in the art of organic chemistry. The preferred GABA analogs tobe utilized in the method of this invention are cyclic amino acids ofFormula I. These are described in U.S. Pat. No. 4,024,175 which isincorporated herein by reference. Another preferred method utilizes theGABA analogs of Formula II, and these are described in U.S. Pat. No.5,563,175 which is incorporated herein by reference.

All that is required to practice the anti-inflammatory method of thisinvention is to administer a GABA analog in an amount that is effectiveto prevent or treat the inflammatory condition. Such anti-inflammatoryamount will generally be from about 1 to about 300 mg per kg of subjectbody weight. Typical doses will be from about 10 to about 5000 mg perday for an adult subject of normal weight.

Pharmaceutical compositions of a GABA analog or its salts are producedby formulating the active compound in dosage unit form with apharmaceutical carrier. Some examples of dosage unit forms are tablets,capsules, pills, powders, aqueous and nonaqueous oral solutions andsuspensions, and parenteral solutions packaged in containers containingeither one or some larger number of dosage units and capable of beingsubdivided into individual doses. Some examples of suitablepharmaceutical carriers, including pharmaceutical diluents, are gelatincapsules; sugars such as lactose and sucrose; starches such as cornstarch and potato starch, cellulose derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose, methyl cellulose, andcellulose acetate phthalate; gelatin; talc; stearic acid; magnesiumstearate; vegetable oils such as peanut oil, cottonseed oil, sesame oil,olive oil, corn oil, and oil of theobroma; propylene glycol, glycerin;sorbitol; polyethylene glycol; water; agar; alginic acid; isotonicsaline, and phosphate buffer solutions; as well as other compatiblesubstances normally used in pharmaceutical formulations. Thecompositions to be employed in the invention can also contain othercomponents such as coloring agents, flavoring agents, and/orpreservatives. These materials, if present, are usually used inrelatively small amounts. The compositions can, if desired, also containother therapeutic agents commonly employed to treat inflammation, forexample, aspirin, naprosyn, and similar anti-inflammatory agents.

The percentage of the active ingredients in the foregoing compositionscan be varied within wide limits, but for practical purposes it ispreferably present in a concentration of at least 10% in a solidcomposition and at least 2% in a primary liquid composition. The mostsatisfactory compositions are those in which a much higher proportion ofthe active ingredient is present, for example, up to about 95%.

Routes of administration of the GABA analog or its salts are oral orparenteral. For example, a useful intravenous dose is between 5 and 50mg, and a useful oral dosage is between 20 and 800 mg. The dosage iswithin the dosing range used in treatment of inflammatory diseases suchas arthritis, or as would be determined by the needs of the patient asdescribed by the physician.

A unit dosage form of the GABA analog to be used in this invention mayalso comprise other compounds useful in the therapy of inflammatorydiseases.

The advantages of using the compounds of Formula I and II, especiallygabapentin and pregabalin, in the instant invention include therelatively nontoxic nature of the compounds, the ease of preparation,the fact that the compounds are well-tolerated, and the ease of IV andoral administration of the drugs. Further, the drugs are not metabolizedin the body.

The subjects as used herein are mammals, including humans.

The ability of GABA analogs to treat inflammatory diseases according tothis invention has been established in several animal models ofinflammation and arthritis.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the effects of pregabalin (designated as S-(+)-3-IBG), itscorresponding R optical enantiomer R-(−)-3-isobutyl GABA (designated asR-(−)-3-IBG), and aCSF (artificial cerebrospinal fluid) on thermal PWL(paw withdrawal latency), on circumference of the knee joint, and ondegree of pain in animals prior to development of acute arthritis.

FIG. 2 shows the effects of 0.9 and 10 mg/mL doses of pregabalin,R-(−)-3-IBG, and aCSF on thermal paw withdrawal latencies, administeredafter development of acute arthritis.

FIG. 3 shows the effects of 0.9 and 10 mg/mL of Pregabalin, R-(−)-3-IBGand aCSF on joint swelling, administered after development of acutearthritis.

FIG. 4 shows the effects of 0.9 and 10 mg/mL of pregabalin, R-(−)-3-IBGand aCSF on pain-related behavior, when administered after developmentof acute arthritis.

The following detailed examples illustrate the specificanti-inflammatory activity of GABA analogs.

EXAMPLE 1

Gabapentin was evaluated in a streptococcal cell wall (SCW)-induced pawedema model. Female Lewis rats were sensitized to SCW (6 μg/rat) in theright tibiotalar joint on Day 0. Vehicle (0.5%hydroxypropylmethylcellulose/0.2% Tween 80) or drug (100 mg/kg, BID) wasadministered orally (10 mL/kg) beginning 1 hour before initiation of thedelayed-type hypersensitivity reaction by systemic SCW (100 μg/rat) onDay 21, and given through Day 24. Assessment of hindpaw edema wasdetermined on Days 22 through 25 by mercury plethysmometry.

Gabapentin was found to significantly inhibit swelling on Days 22, 23,24, and 25 (58%, 77%, 83%, and 81%, respectively).

EXAMPLE 2

Pregabalin was evaluated in a similar assay and showed dramaticanti-inflammatory activity. The assay is a streptococcal cell wall (SCW)induced reactivation arthritis assay. Female Lewis rats were injectedintra-articularly with 10 μL of 100 p fraction peptidoglyeanpolysaccharide (PG-PS) suspended in phosphate buffered saline (PBS). Thecontralateral joints were injected with PBS as control. Systemicchallenge with 100 μg of PG-PS was given via the tail vein 21 days afterthe initial inoculation. The animals were dosed orally three times a daywith pregabalin (3, 10, and 30 mg/kg) on a 12-hour cycle for 72 hours.The first dose was given 1 hour before the systemic challenge.

Systemic challenge on Day 21 of previously sensitized animals with SCWresulted in acute swelling in the sensitized ankle. The volume of theankles increased by about 0.5 mL within 72 hours. Pregabalin at 10 and30 mg/kg dose dependently attenuated the increase in edema up to 40%during the 72-hour observation period. The results are present in Table1.

TABLE 1 Effect of Pregabalin on Ankle Swelling (Days 0-20 weresensitization period) Swelling (delta edema, mL) Day 21 PG-PS PregabalinChallenge Day 22 Day 23 Day 24 oral (mg/kg) 0 0.20 0.45 0.50 0 (n = 6) 00.12 0.38 0.50 3.0 (n = 6) 0 0.04 0.25 0.31 10.0 (n = 4) 0 0.06 0.200.21 30.0 (n = 6)

The foregoing assay establishes that GABA analogs such as gabapentin andpregabalin are effective anti-inflammatory agents and cause a reductionin swelling of the type encountered in patients suffering fromarthritis.

EXAMPLE 3

Pregabalin (which is an S-isomer, and is also known as CI-1008, and asS-(+)-3-IBG) was also evaluated in the following anti-inflammatory test,along with the corresponding R-isomer,(R)-3-(aminomethyl)-5-methyl-hexanoic acid (also referred to asR-(−)-3-IBG). Acute experimental arthritis was induced in rats byinjection of kaolin and carrageenan into the knee joint. Theinflammatory agent, carrageenan, causes plasma extravasation and edemafollowing the release of neuropeptides and other inflammatory mediatorsinto the joint cavity. Concomitant with the injury to the joint tissue,both peripheral and central sensitization occurs, which is manifested inthe awake rat as hyperalgesia, which can be easily quantified bymeasuring a reduction in paw withdrawal latencies to a radiant heatsource. Both pregabalin and its R-stereoisomer (R-(−)-3-IBG) wereadministered before inflammation was induced, and after the inflammationwas developed.

Thirty-six male Sprague-Dawley rats (235-380 g) were anesthetized withsodium pentobarbital (Nembutal; 50 mg/kg⁻¹ i.p.). A microdialysis fiber(200 μm o.d., 45000 MW Cut-off, Hospal AN69) was coated with epoxyresin, except for a 2 mm section. A small midline incision was made inthe back at the level of the last rib. The muscle was then removed fromaround the T₁₂ vertebra and a hole drilled in both lateral aspects. Themicrodialysis fiber was then passed transversely through the dorsal hornof the spinal cord between lumbar segments L₄-L₆ so that the permeable 2mm of the fiber lay in the dorsal horn. The microdialysis fiber wasconnected to PE₂₀ tubing (Becton and Dickson) which was then tunneledunder the skin to the nape of the neck. The fiber was stabilized withdental cement. Artificial cerebrospinal fluid (aCSF) was pumped throughthe tubing at a rate of 5 μL/min for 1 hour before the PE₂₀ was sealedand the animals allowed to recover.

As a measure of thermal hyperalgesia, animals were tested for pawwithdrawal to radiant heat. On the day following fiber placement,animals were housed in small lucite cubicles on an elevated glass plate.Radiant heat was applied to the plantar surface of the heel of thehindpaw until the rat lifted the paw. The time at which this occurredwas considered the paw withdrawal latency (PWL). Both paws were testedindependently at 5-minute intervals, for a total of 5 trials. A mean ofthese five readings was used as the PWL. In pre-treatment rats, PWL wasmeasured before administration of any GABA analogs (baseline) and afterthe GABA analog had been infused for 1.5 hours, at which time kaolin andcarrageenan was injected into the knee joint. PWL was measured for afinal time 4 hours after arthritis induction. In the post-treatmentgroup, the animals were tested before induction of arthritis in the kneejoint (control), 4-hours post-induction, and 1.5 hours after of druginfusion, i.e. 5.5 hours after arthritis induction. A decrease in thePWL to radiant heat in an animal with knee joint inflammation isindicative of secondary hyperalgesia.

The circumference of the knee joint was also measured before injectionof kaolin and carrageenan (control) using a flexible tape measure. Theextent of guarding of the hindpaw was also noted after arthritis wasinduced. To quantify these changes, the animals were graded by asubjective pain rating scale (0-5), where: 0 is normal; 1 is curling ofthe toes; 2 is eversion of the paw; 3 is partial weight bearing; 4 isnon-weight bearing and guarding; and 5 is avoidance of any contact withthe hindlimb.

Induction of Arthritis

Rats were anesthetized briefly with sodium methohexital (Brevital; 60mg/kg⁻¹ i.p.) after the control behavioral test (post-treatment group)or after infusion of the drug (pre-treatment group). The knee joint wasthen injected with 3% kaolin and 3% carrageenan suspended in sterilesaline (0.1 mL; pH 7.4). The knee joint was then flexed manually untilthe rat awoke (approximately 5 minutes).

Administration of GABA Analogs

All GABA analogs were dissolved in an artificial cerebral spinal fluidsolution (aCSF) (pH 7.4, adjusted by bubbling with 95% CO₂/5% O₂) andinfused through the spinal cord at 5 μL/min⁻¹. The animals receivedeither pregabalin, R-(−)-3-IBG, or aCSF. In the post-treatment group,the GABA analogs were infused at concentrations of 0.1, 0.9, and 10mg/mL. In contrast, the pre-treatment group received a single dose of 10mg/mL.

Statistical Analysis

The data was normally distributed. Statistical analyses were carried outusing unpaired t-tests for comparison of differences between treatmentgroups at the same timepoint. Paired t-tests were used to compare beforeand after treatment within the same group. A P value of less than 0.05was used to indicate significance. Data are expressed as means± s.e.m.Tests were carried out using Statistica (Jandel Corporation).

Results

Effect of Pregabalin and its R-Isomer Infused Into the Spinal CordBefore the Development of Acute Arthritis

Infusion of 10 mg/mL of pregabalin, its R-isomer, or aCSF into thedorsal horn of the spinal cord alone did not change PWL in the thermalhyperalgesia test when compared to baseline values. The PWL of the ratstreated with aCSF before the induction of inflammation was significantlyreduced at 4 hours after injection of kaolin and carrageenan (P<0.01,paired t-test), when compared to the value recorded immediately beforeinjection. There was also a significant difference (P<0.05, unpairedt-test) between the injected limb and the uninjected limb at this time.

However, in the rats infused with a concentration of 10 mg/mL pregabalinor its R-isomer through the spinal cord for 1.5 hours before theinjection of kaolin and carrageenan into the knee joint, no secondarythermal hyperalgesia was observed 4-hours post-injection (FIG. 1, toppanel). No significant difference was observed between the PWL valuerecorded 4 hours after inflammation and that recorded prior to injectionof kaolin and carrageenan, nor between the inflamed limb and theuninflamed limb 4 hours after injection of kaolin and carrageenan.

Infusion of pregabalin or its R-isomer into the spinal cord for 1.5hours before the induction of arthritis also significantly reduced(P<0.05; unpaired t-test) the amount of swelling typical after injectionof kaolin and carrageenan into the knee joint by approximately 30%, whencompared to rats in which aCSF was infused (FIG. 1, middle panel).Further, pre-treatment with pregabalin or its R-isomer prevented thedevelopment of abnormal paw posture indicative of spontaneous pain (FIG.1, bottom panel).

Effect of Pregabalin and its R-isomer Infused Into the Spinal Cord Afterthe Development of Acute Arthritis

Four hours after the induction of acute inflammation of the knee joint,there was a decrease in the PWL to radiant heat of the ipsilateralfootpad, when compared to the control value, in all animals tested(n=30), indicating the presence of secondary hyperalgesia (FIG. 2). Thisdecrease was significant (paired t-test, p<0.01). Four hours afterinflammation of the knee joint, there was a significant increase in kneejoint circumference compared to the measurement recorded immediatelybefore injection of kaolin and carrageenan (P<0.05, paired t-test; FIG.3). After inflammation, there was also a change in the rats' posture(decreased weight bearing upon the swollen limb, and curling of thetoes) reflected by the increased spontaneous pain rating score given tothe rats (FIG. 4, hollow bars).

The infusion of 0.9 mg/mL pregabalin or its R-isomer into the dorsalhorn of the spinal cord reduced the thermal hyperalgesia at 5.5 hours(FIG. 2, upper panel). Although the PWL recorded after infusion ofeither drug was significantly different from that recorded 4 hours afterinflammation, it was still significantly less than the control value.Pregabalin was more effective in reducing thermal hyperalgesia than itsR-isomer. Infusion of a higher dose, 10 mg/mL, of pregabalin or itsR-isomer, after inflammation of the knee joint, resulted in a return ofthe PWL to the control value (FIG. 2, lower panel). In contrast,infusion of aCSF into the dorsal horn did not reduce the thermalhyperalgesia; the PWL at 4 hours after inflammation and after aCSFinfusion were not significantly different.

The spontaneous pain was also reduced by infusion of both doses ofpregabalin and its R-isomer. After infusion of either isomer of thedrug, the paw posture was almost normal, whereas after infusion of aCSF,curling of the toes and eversion of the paw were observed.

The results from these studies show that injection of kaolin andcarrageenan into the knee joint of the rat results in an acute arthritiswhich is characterized by secondary thermal hyperalgesia, swelling ofthe knee joint, and spontaneous pain. Infusion of pregabalin andR-(−)-3-IBG into the dorsal horn of the spinal cord for 1.5 hours beforethe injection of kaolin and carrageenan reduced the amount of swellingobserved, and blocked the secondary hyperalgesia and spontaneous pain.The GABA analogs are thus useful to treat inflammatory diseases,especially arthritis.

EXAMPLE 4

Gabapentin, another GABA analog, was evaluated in a similar assay andshown to be effective in both preventing and reversing the affects ofkaolin/carrageenan knee joint inflammation, secondary heat hyperalgesiaand spontaneous pain-related behaviors.

Methods

Thirty animals in two experimental groups were treated (1) prior to, and(2) after induction of experimental arthritis. Inflammation was inducedwithin the knee joint by injection of kaolin/carrageenan. Gabapentin oraCSF was administered through a microdialysis fiber positioned in thedorsal horn for spinal treatment, or subcutaneously in the nape of theneck for systemic release. All experiments were carried out by anobserver blind to the drug treatment.

Placement of microdialysis fibers. Sprague-Dawley rats (220-270 g) wereanesthetized with sodium pentobarbital (nembutal, 50 mg/kg, i.p.). Amicrodialysis fiber (200 μm o.d., 45000 MW Cut-off, Hospal AN69) wascoated with epoxy resin, except for a 2 mm section. In 24 animals, themicrodialysis fiber was placed in the dorsal horn. A small midlineincision was made in the skin over the L₁ vertebral level. The L₁vertebra was cleared of muscle and a hole drilled in both sides of thelamina. The microdialysis fiber was then passed through the holes in thevertebrae and transversely through the dorsal horn of the spinal cord.The microdialysis fiber lay between L₄-L₆ segments with the permeable 2mm of the fiber in the dorsal horn. The microdialysis fiber wasconnected to PE₂₀ tubing (Becton Dickinson) which was tunneled under theskin to the nape of the neck. The connecting joint between themicrodialysis fiber and PE₂₀ tubing was stabilized with dental cement.The aCSF was pumped through the tubing at a rate of 5 μL/min for 1 hourbefore the PE₂₀ tubing was sealed, and the animal was allowed to recoverfor 24 hours. Once the rats were awake, they were examined for motordeficits; any rat which had motor deficits was excluded from the study.As a systemic control for drug administration in another 6 rats, themicrodialysis fiber was implanted in the subcutaneous tissue at the napeof the neck.Behavior testing and assessment of arthritis. The PWL to noxious radiantheat was tested as a measure of thermal hyperalgesia. A decrease in thePWL in animals with knee joint inflammation was interpreted asindicative of secondary hyperalgesia. Since the radiant heat stimulus isapplied to the plantar surface of the hindpaw at quite some distancefrom the inflamed knee joint, the measure reported represents secondaryheat hyperalgesia.

On the day following fiber placement, animals were housed in smalllucite cubicles on an elevated glass plate. Radiant heat was applied tothe plantar surface of the hindpaw until the rat lifted the paw. Thetime at which this occurred was considered the PWL. Both paws weretested independently at 5-minute intervals for a total of five trials. Amean of these five readings was used as the PWL for each time points. Inpretreatment rats (n=12), PWL was measured before administration of anydrugs (baseline), after the drug had been infused for 1.5 hours(post-drug), and 4 hours after arthritis. In the post-treatment group(n=18), the animals were tested before induction of arthritis in theknee joint (baseline), 4 hours after induction of arthritis, and 1.5hours after drug infusion, i.e., 5.5 hours after arthritis induction.

The pain-related behavior, the extent of guarding of the hindpaw of thearthritis limb, was scored by two independent observers. To quantifythese changes, the animals were graded by a subjective pain rating scale(0-5) where: 0 is normal, 1 is curling of the toes, 2 is eversion of thepaw, 3 is partial weight bearing, 4 is non-weight bearing and guarding,and 5 is avoidance of any contact with the hindpaw.

The circumference of the knee joint was also measured using a flexibletape measure before induction of arthritis (baseline), 4 hours afterinduction of arthritis (pretreatment and posttreatment group), and 1.5hours after drug infusion in the posttreatment group (5.5 hours afterinduction of arthritis).

Induction of arthritis. Rats were anesthetized briefly with methohexitalsodium (Brevital sodium, 60 mg/kg i.p.) after baseline behavior test(post-treatment group) or after infusion of the drug (pre-treatmentgroup). The knee joint was then injected with 0.1 mL of 3% kaolin and 3%carrageenan suspended in sterile saline, and was flexed manually untilthe rat awoke (approx. 5-10 min.).Administration of drug. The animal received either gabapentin or aCSF asa control. The gabapentin was dissolved in aCSF. Both gabapentin andaCSF were infused through the microdialysis fiber at a rate of 5μL/minute. The pH of the gabapentin solution and aCSF were adjusted bybubbling with 95% CO₂/5% O₂ (about 7.4) before using.

The single dose of 10 mg/mL of gabapentin was used for the study.

Statistical analysis. The results for each group were expressed as theaverage percent change from baseline ± the standard error of the mean(s.e.m.). Paired t-tests were used to compare each animal's testresponses to its own baseline (P<0.01).

Results

Baseline measures. The baseline PWL, spontaneous behavior, and kneejoint circumference of all rats used in these studies were measuredprior to infusion of the drug or vehicle through the spinal cord orsubcutaneously (Table 2). The mean PWL and knee joint circumference were10.52±0.39 sec and 5.26±0.03 cm, respectively. No spontaneouspain-related behaviors were noted and a score of zero given.Consequent changes with joint inflammation. In Table 2, the expectedoutcome in arthritic animals for all measures is presented. The dataincludes the combined measures for the aCSF arthritic control animalsfrom both treatment groups. In the aCSF-treated arthritic control rats(n=12), 4 hours after injection of kaolin and carrageenan, the PWL tonoxious radiant heat decreased to 76% of baseline value. This decreasewas significant (paired t-test, p<0.01) and indicated the presence ofsecondary hyperalgesia.

In arthritic animals, there was a significant change in the hindpawposture of the rat, indicative of spontaneous ongoing pain-relatedbehavior. These postural changes, representing spontaneous ongoingpain-related behavior, were represented by a score of 1.25±0.13(p<0.01). A significant 14% increase in knee joint circumference isnoted compared to the baseline (paired t-test, p<0.01).

TABLE 2 Non-arthritic Vs. Arthritic Animals PWL PWL (% of BehaviorCircumference Circumference (sec.) baseline) Score (cm) (% of baseline)Baseline 11.47 ± 0.56 100 0 5.18 ± 0.04  100 Arthritis (4 h)  8.66 ±0.56* 76.42 ± 3.10* 1.25 ± 0.13* 5.92 + 0.09* 114.38 ± 1.86* *p < 0.01.The effect of gabapentin infusion directly into the spinal cord beforeknee joint inflammation. Gabapentin was effective in preventing thedevelopment of secondary hyperalgesia responses to the applied radiantheat. Gabapentin or aCSF were infused through the microdialysis fiberinto the spinal cord before the knee joint was injected with kaolin andcarrageenan. After 1.5 hours of spinal drug infusion, there were nosignificant changes of the PWL to the radiant heat compared to thebaseline (Table 3). Four hours after injection of the knee joint withkaolin and carrageenan, the PWL response to radiant heat and the postureof the hindpaw with arthritis were not significantly changed fromnon-arthritic baseline. In contrast, the aCSF-treated animals had asignificant reduction in their PWL responses, and demonstratedsignificant spontaneous pain-related behaviors. The circumference of theinflamed joint was increased significantly 4 hours after arthritis,similar to the aCSF arthritic control rats. Thus, gabapentin was highlyeffective in preventing the development of secondary heat hyperalgesiaand measures of spontaneous pain-related behaviors.

TABLE 3 Effects of Gabapentin Administered Prior to InflammationBaseline PWL PWL Behavior Score Circumference (% of (1.5 h after (4 hafter joint (4 h after joint (4 h after joint Groups control) druginfusion) injection) inspection) injection) Gabapentin 100 105.18 ± 4.56100.03 ± 4.37 0.67 ± 0.20 114.20 ± 1.53* (n = 6) aCSF 100  93.12 ± 6.31 74.47 ± 3.44* 1.33 ± 0.2* 114.87 ± 1.74* (n = 6) *p < 0.01.Effect of gabapentin infusion into the spinal cord or subcutaneouslyafter knee joint inflammation. Post-treatment of arthritic animals withgabapentin reversed the secondary heat hyperalgesia and spontaneouspain-related behaviors when administered spinally. Two groups of animalsreceived gabapentin in post-treatment studies (Table 4). One group ofrats was infused with the drug through a microdialysis fiber implanteddirectly into the spinal cord; the other group received gabapentinsystemically through a microdialysis fiber implanted subcutaneously atthe nape of the neck.

Four hours after injection of kaolin and carrageenan, all animalsdisplayed reduced PWL responses and spontaneous pain-related behaviors.In the group infused with gabapentin spinally, the PWL significantlydecreased to about 81% of baseline measurements (paired t-test, p<0.01). By 1.5 hours after spinal gabapentin infusion, the PWLmeasurements returned back to the baseline, and the toes became almostflat.

TABLE 4 Effects of Gabapentin Administered After Inflammation PWL orBehavior Circumference PWL PWL Score Circumference (% of (after 4 h(after 5.5 h (after 5.5 h (after 5.5 h Groups control) arthritis)arthritis) arthritis) arthritis) Gabapentin 100 80.71 ± 3.23* 100.85 ±10.63 0.50 ± 0.20  122.22 ± 2.32* (spinal cord) (n = 6) Gabapentin 10085.05 ± 3.68*  81.89 ± 4.43* 1.17 ± 0.29* 120.66 ± 3.59* (subcutaneous)(n = 6) aCSF 100 78.37 ± 5.37*  78.57 ± 4.38* 1.17 ± 0.28* 113.89 ±3.49* (spinal cord) (n = 6) *p < 0.01.

In the group which was infused with gabapentin subcutaneously, the PWLto noxious radiant heat significantly decreased by 15% from baselinemeasurements 4 hours after joint injection, and after 1.5 hours druginfusion, the PWL continued to decrease to 82% of the baseline value,similar to aCSF control arthritic rats. Both the pain-related behaviorscore and the circumference of the inflamed joint increasedsignificantly after 4 hours arthritis and 1.5 hours drug infusion (5.5 hpost) for all groups.

The foregoing study establishes that GABA analogs such as gabapentin areeffective in both preventing and reversing the affects ofkaolin/carrageenan knee joint inflammation on secondary heathyperalgesia and spontaneous pain-related behaviors. In both treatmentgroups, the significant finding was the ability of gabapentin to retain(or return) the PWL latency scores to baseline. Its effectiveness inreducing the hyperalgesia and pain-related behavior after the arthritisis fully developed in this model indicates that gabapentin and similarGABA analogs will have clinically useful effects in clinicalinflammatory conditions.

1. A method for treating inflammatory diseases comprising administeringto a subject in need of treatment an effective anti-inflammatory amountof a compound of formula I:

wherein R₁ is hydrogen or lower alkyl and n is an integer of from 4 to6, or a pharmaceutically acceptable salt thereof.
 2. The method of claim1, wherein said compound is 1-(aminomethyl)-cyclohexane acetic acid. 3.The method of claim 2, wherein the inflammatory disease is arthritis. 4.The method of claim 3, wherein the inflammatory disease is rheumatoidarthritis.
 5. The method of claim 2, wherein the effectiveanti-inflammatory amount of a compound of formula I is administered inan oral dosage form.
 6. The method of claim 5, wherein the oral dosageform is a tablet, capsule or pill.
 7. The method of claim 6, wherein theoral dosage form comprises between 20 and 800 mg of1-(aminomethyl)-cyclohexane acetic acid.
 8. A method for treatinginflammatory diseases comprising administering to a subject in need oftreatment an effective anti-inflammatory amount of a compound of formulaII:

wherein R₁ is a straight or branched alkyl of from 1 to 6 carbon atoms,phenyl, or cycloalkyl of from 3 to 6 carbon atoms; R₂ is hydrogen ormethyl; and R₃ is hydrogen, methyl, or carboxyl, or a pharmaceuticallyacceptable salt thereof.
 9. The method of claim 8, wherein said compoundis (S)-3-(aminomethyl)-5-methylhexanoic acid.
 10. The method of claim 9,wherein the inflammatory disease is arthritis.
 11. The method of claim10, wherein the inflammatory disease is rheumatoid arthritis.
 12. Themethod of claim 9, wherein the effective anti-inflammatory amount of acompound of formula II is administered in an oral dosage form.
 13. Themethod of claim 12, wherein the oral dosage form is a tablet, capsule orpill.
 14. The method of claim 13, wherein the oral dosage form comprisesbetween 20 and 800 mg of (S)-3-(aminomethyl)-5-methylhexanoic acid.